A novel autophagy activator ginsenoside Rh2 enhances the efficacy of immunogenic chemotherapy

schematic model illustrating the effects of G-Rh2 in enhancing MTX-induced immunogenic cell death (ICD) and promoting its anti-tumour effects via reprogramming the tumour microenvironment. G-Rh2 enhances MTX-induced hallmarks of ICD, such as ATP release, cell surface calreticulin (CALR) exposure and HMGB1 (high mobility group box 1) release. Mechanistically, G-Rh2 promotes MTX-induced ATP release via transcription factor EB (TFEB)/transcription factor E3

Dear Editor, Immunogenic cell death (ICD) caused by certain chemotherapeutic drugs, including mitoxantrone (MTX), elicits specific protective anti-tumour immunity and is, thus, regarded as an effective strategy for cancer treatment. Pharmacological enhancement of autophagy is effective in enhancing anticancer immune responses to ICD-inducing chemotherapeutic drugs. Here, we discover that ginsenoside Rh2 (G-Rh2) enhance MTX-induced hallmarks of ICD, which include increased ATP release, relocation of calreticulin (CALR) to the cell membrane and HMGB1 (high mobility group box 1) secretion. Mechanistic studies reveal that G-Rh2induces autophagy through the activation of TFEB (transcription factor EB) and TFE3 (transcription factor E3), which contributes to the synergistic effect of G-Rh2 and MTX on promoting ATP release. In addition, G-Rh2 increased endoplasmic reticulum (ER) stress with phosphorylated eukaryotic initiation factor eIF2α, which promoted MTX-induced cell surface calcineurin exposure. Consequently, G-Rh2 enhanced the in vivo anti-tumour effect of MTX in immunocompetent mice bearing MCA205 tumour with increased cytotoxic T lymphocytes (CTLs). Thus, G-Rh2 represents a promising drug candidate for treating cancers in combination with ICD-inducing chemoimmunotherapeutic drugs such as MTX.
In response to certain cellular stimuli, injured or stressed cells release DAMPs on their surface to produce immunostimulatory effects, including recruiting and activating immune cells that ultimately kill cancer cells. 1 This kind of regulated cell death is referred to as ICD. 1 capture antigens and trigger tumour-specific cytotoxic Tcell responses. Extracellular HMGB1 binds to its receptor such as TLR4 on DCs, which promotes tumour antigen processing and presentation to T cells. Thus, the induction of ICD triggers long-lasting anti-tumour immunity, and it is regarded as an effective strategy for cancer treatment. 2,3 TFEB and TFE3 are key transcription factors that regulate autophagy. 4,5 With respect to ICD, the activation of several stress pathways, including autophagy, is indispensable for intracellular ATP release. 6 Induction of autophagy by several ICD inducers enhances the anticancer effects via modulating the tumour microenvironment. 7 Therefore, autophagy activation to enhance the effects of chemotherapeutics on inducing ICD holds promise for anticancer therapy. 8 Driven by these considerations, we sought to identify novel autophagy enhancer(s) and evaluate their roles in stimulating anticancer immunity in combination with ICD-inducing chemotherapeutics in U2OS cells (human bone osteosarcoma epithelial cells), MCA205 cells (mouse fibrosarcoma cells) and MCA205-inoculated immunocompetent mice.
We further determined whether G-Rh2 induces hallmarks of ICD with or without a low concentration of F I G U R E 1 Ginsenoside Rh2 (G-Rh2) induces autophagy via transcription factor EB (TFEB) and transcription factor E3 (TFE3) activation: (A) G-Rh2 increases LC3-II levels. U2OS cells were exposed to different doses of G-Rh2 (1, 5 and 10 μM) for 16 h, and LC3-II was measured; (B and C) G-Rh2 induces autophagic flux. U2OS cells were exposed to G-Rh2 (10 μM) with or without CQ (50 μM, added at last for 3 h) for 16 h, LC3-II was measured (B) and quantified by ImageJ (C); (D and E) G-Rh2 increases LC3 puncta. After treating U2OS cells transiently expressing GFP-LC3 with G-Rh2 for 16 h, LC3 puncta was visualized (D) and quantified (E); (F and G) G-Rh2 increases autolysosomes. After treating U2OS cells transiently expressing GFP-RFP-LC3 with G-Rh2 (10 μM) for 16 h, LC3 puncta was recorded (F) and red-only puncta (autolysosome) was quantified (G). Scale bar: 15 μm; (H and I) G-Rh2 induces the relocation of TFEB and TFE3 from the cytoplasm into the nucleus. U2OS cells transiently expressing 3XFlag-TFEB or GFP-N1-TFE3 were incubated with indicated doses of G-Rh2 (1, 5 and 10 μM) for 16 h. The distribution of TFEB in cells was detected by fluorescence microscope. Scale bar: 15 μm. *p < .05; **p < .01 F I G U R E 2 Ginsenoside Rh2 (G-Rh2) enhances autophagy-dependent ATP release: (A) G-Rh2 reduces intracellular ATP contents. U2OS cells were exposed to vehicle control, G-Rh2 (10 μM), a low dose of MTX low (1 μM) or their combination for 16 h. The intracellular ATP contents were examined by quinacrine staining. MTX high (5 μM) was used as a positive control. Scale bar: 15 μm; (B) quantification data in (A) MTX (MTX low ). G-Rh2 or MTX slightly but significantly reduced intracellular ATP release, and G-Rh2 combined with MTX low substantially reduced the intracellular ATP contents (Figure 2A,B). Autophagy deficiency by knocking down ATG5 ( Figure 2C-E) attenuated G-Rh2 plus MTX lowinduced decrease in intracellular ATP contents as reflected by quinacrine staining (Figure 2F) 9 and the release of extracellular ATP contents ( Figure 2G). Similarly, the combination of G-Rh2 and MTX-induced decrease of intracellular ATP and increase of extracellular ATP was inhibited in TFE3-and TFEB-knocked-down cells ( Figure 2H,I). These findings demonstrate that the synergistic effect of G-Rh2 and MTX on ATP release depends on autophagy induction.
Furthermore, G-Rh2 increased MTX low -induced cell surface exposure of CALR as reflected by immunostaining and flow cytometry analysis ( Figures 3A-C and S2A,B). The combination of G-Rh2 and MTX low also increased an HMGB1 release ( Figure 3D-G). To determine how G-Rh2 and MTX induce cell surface CALR exposure, we next showed that G-Rh2 increased ER stress, especially PERK/p-eIF2α/ATF4 axis ( Figure S3A-H). We discovered that PERK knock-down reduced G-Rh2-induced ER stress ( Figure S3I-K) and comprised G-Rh2 plus MTX-caused cell surface CALR exposure ( Figure 3H,I). Interestingly, the inhibition of ER stress by 4-PBA (4-phenylbutyric acid) also attenuated cell surface relocation of CALR ( Figure  S3L). These results indicate that ER stress is indispensable for the role of G-Rh2 in enhancing MTX-induced cell surface relocation of CALR. Apart from ICD, the combination of G-Rh2 and MTX also induced cell apoptosis, and this effect was further enhanced by the lysosomal inhibitor CQ ( Figure S4A,B), suggesting that the apoptosis may also be involved in anticancer effects. To understand the crosstalk of autophagy and ER stress during ICD, we found that the inhibition of lysosomal functions by CQ did not further enhance ER stress ( Figure S4C), and ER stress inhibitor 4-PBA attenuated autophagy in response to G-Rh2 ( Figure S4D). Consistently, CQ did not enhance G-Rh2 plus MTX-induced cell surface CALR exposure ( Figure  S4E) but attenuated G-Rh2 plus MTX-induced ATP release ( Figure S4F), supporting a critical role of autophagy in promoting ATP release. Furthermore, though apoptosis inhibitor Z-VAD-FMK inhibits G-Rh2 plus MTX-induced apoptosis ( Figure S5B), Z-VAD-FMK did not inhibit G-Rh2 plus reduction of intracellular ATP levels ( Figure S5A), and cell surface CALR exposure ( Figure S5C,D), further strengthen the hypothesis that ICD rather than apoptosis is involved in the anti-tumour effect of G-Rh2 plus MTX.
To confirm the conserved synergistic effects of G-Rh2 and MTX in enhancing ICD, we showed that in immunosurveillance MCA205 mouse fibrosarcoma cells, G-Rh2 also enhanced autophagy ( Figure S6A), induced ER stress ( Figure S6B,C). Consistently, G-Rh2 enhanced MTX lowinduced cell surface CALR exposure, HMGB1 release from the nucleus, and extracellular ATP release ( Figure S6D-I), suggesting that G-Rh2 also promotes MTX-induced ICD in MCA205 fibrosarcoma cells. MCA205 cells inoculated in mice are well characterized as a suitable model for the investigation of immune response, and the tumour infiltration on the skin can also be considered to be orthotopic. 10 We next determined the synergistic anti-tumour role of G-Rh2 in combination with MTX by inoculating MCA205 cells into immunocompetent C57 mice followed by drug treatment as shown in the schematic model ( Figure 4A). We showed that G-Rh2, MTX and a combination of G-Rh2 and MTX did not affect mice's body weight ( Figure 4B), but the combination treatment significantly mitigated tumour growth ( Figure 4C,D). Importantly, the combination treatment increased the abundance of CTLs while exerting minimal effect on that of regulatory T cells (Tregs) (Figures 4E,F and S7). Consequently, this combination treatment increased the CTL/Treg ratio ( Figure 4G), suggesting that G-Rh2 and MTX synergistically promote anti-tumour immunity by tipping the immune balance and reprogramming the tumour microenvironment.
Overall, this study illustrates that G-Rh2 is responsible for TFE3/TFEB-mediated autophagy activation and ER-stress induction with phosphorylated eIF2α, and it synergizes with immunogenic chemotherapeutic drug MTX to enhance MTX-induced ICD, which consequently facilitates the anti-tumour effect of MTX in immunocompetent shows that G-Rh2 promotes mitoxantrone (MTX)-induced reduction of intracellular ATP contents; (C-E) after transfected U2OS cells with siRNA to knock down the expression of key autophagy gene ATG5, ATG5 and LC3-II levels were measured (C) and quantified (D and E); (F and G) the inhibition of autophagy through knocking down of the expression of ATG5 attenuates G-Rh2 plus MTX-induced ATP release. After ATG5 knocking down, U2OS cells were treated with vehicle control, G-Rh2 (10 μM), MTX low (1 μM) or the combination of G-Rh2 (10 μM) and MTX low (1 μM) for 16 h, the intracellular ATP levels were measured by quinacrine staining (F) and the extracellular ATP contents were measured by a bioluminescent assay kit (G); (H and I) inhibition of autophagy by transcription factor EB (TFEB)/transcription factor E3 (TFE3) knockdown attenuates G-Rh2 plus MTX-induced ATP release. After TFE3 and TFEB knockdown, U2OS cells were incubated with G-Rh2, MTX low , or the combination of G-Rh2 (10 μM) and MTX low (1 μM) for 16 h, and the intracellular ATP contents were measured by quinacrine staining (H), and the extracellular ATP contents were measured by a bioluminescent assay kit (I). Scale bar: 15 μm. *p < .05, **p < .01  Figure 4G). Our findings provide mechanistic insights into how G-Rh2 synergizes with MTX to amplify its effects on ICD induction and anti-tumour activity and provide a novel link between G-Rh2-activated TFEB/TFE3-dependent autophagy induction and ICDinvolved anti-tumour effect. Our discovery indicates that G-Rh2 is a novel drug candidate for improving the antitumour effects of immunogenic chemotherapies.

C O N F L I C T S O F I N T E R E S T
There are no conflicts of interest between all authors.